The work described herein was supported, in part, by Grant PHS GM 27314 from the U.S. Public Health Service, Institute of General Medical Sciences.
This invention relates to the field of recombinant DNA. In one of its aspects, this invention relates to a DNA fragment which inhibits degradation of abnormal proteins in bacterial cells. In another aspect, this invention relates to a method of isolating this stabilizing DNA fragment from T-even phage particles. In another aspect, this invention relates to a plasmid containing the stabilizing DNA fragment. In yet another aspect, this invention relates to a method of amplifying foreign proteins encoded by cloned DNA by including the stabilizing DNA fragment into the genetic machinery of bacterial host cells.
Most living cells possess efficient systems for recognizing and eliminating abnormal proteins. As used herein, the term "abnormal proteins" refers to proteins with abnormal conformations, protein fragments, polypeptide sequences containing amino acid analogues, missense mutant proteins, nonsense protein fragments, proteins encoded by cloned DNA, and other polypeptides not ordinarily present in healthy, viable cells. In E. coli cells, for example, the half-lives of protein fragments and protein with abnormal conformations are much shorter than the half-lives of normal proteins. About 3% of normal proteins turns over each hour in E. coli cells. Abnormal proteins, however, are so short-lived that they are found in much lower quantities than normal proteins or fail to accumulate to detectable levels.
Little is known about the mechanism by which living cells detect and degrade abnormal proteins. Some information is available about this system in E. coli cells. See, for example, Simon et al., Nature, 275, 424 (1978). It is known that the degradation of abnormal proteins in E. coli cells is ATP-dependent. If cellular ATP levels are substantially reduced, the turnover of abnormal proteins and protein fragments is also reduced. It is also believed that the ATP-dependent degradative mechanism is bound to the E. coli cell membrane. However, the identities and functions of the enzymes responsible for recognizing and degrading abnormal proteins remain obscure in E. coli and in other bacterial cells.
The degradation systems possessed by living cells present a major obstacle to the manufacture of useful proteins by means of recombinant DNA or molecular cloning procedures. By these procedures, the genes which code for eukaryotic proteins are introduced into host cells, such as bacterial cells, which then express the foreign genes as the eukaryotic proteins. The recombinant DNA procedures involve isolating the relevant genes, inserting them into suitable cloning vehicles, such as bacterial plasmids or viruses, and transforming host cells by introducing the hybrid cloning vehicles into the cells. The transformed cells which contain the relevant genes are then selected from all the other cells and are grown in cultures. By such techniques, bacterial cells have been induced thus far to manufacture human growth hormone, human insulin, human interferon, etc. However, the bacterial cells apparently recognize these products as abnormal proteins, and under most circumstances, degrade them. Thus, the ability of living cells to detect and degrade abnormal proteins may severely limit the yields of useful proteins obtained from cloned cells.
To increase such yields, it would be highly desirable to provide a method for inhibiting or suppressing the degradation mechanism in cells carrying cloned genes. Such a method, when combined with already known recombinant DNA methods, would provide a highly efficient means for producing useful proteins.
It has previously been reported that the bacteriophage known as T4 influences the degradation function in E. coli cells. Simon et al., in Nature, supra, reported that T4 infection of E. coli cells inhibits degradation of abnormal polypeptide sequences but does not affect the turnover rate of normal bacterial proteins. In the studies reported therein, it was found that adsorption of T4 particles to the E. coli surface and injection of T4 DNA were not by themselves sufficient to alter protein turnover. It was found that inhibition of degradation requires the synthesis of early T4 proteins. It was also reported therein that other phages, such as T5 and T7, also inhibit degradation in E. coli cells but not as to great an extent as does T4 phage. However, infecting E. coli cells with T4, T5 or T7, phage particles is fatal to the host cells, and this method is unsuitable for amplifying proteins expressed by cloned DNA.
In order to maximize the yields of eukaryotic proteins produced by recombinant DNA techniques, it would be highly desirable to excise from T4 phage particles the gene(s) responsible for inhibiting the degradation mechanism and to insert the same gene(s) into bacterial host cells. In this manner, the degradation mechanism of cells carrying cloned DNA can be turned off without killing the cells as by infection with T4 phage particles.
It would thus be desirable to isolate a DNA fragment from T4 phage particles which will inhibit the degradation of abnormal proteins in bacterial host cells.
It would also be desirable to produce a cloning vehicle, such as a bacterial plasmid, which contains the stabilizing T4 DNA fragment.
It would also be desirable to transform bacterial cells by introducing the stabilizing T4 DNA fragment.
It would further be desirable to produce transformants which contain the genes for producing eukaryotic proteins as well as the stabilizing T4 DNA fragment.